Global DNA hypomethylation and regional DNA hypermethylation are known to accompany the development and progression of breast cancer. We hypothesize that global and gene-specific DNA methylation are associated with breast cancer risk. We further hypothesize that folate, vitamin B12 and vitamin C status might influence breast cancer risk via effects on DNA methylation. The rationale for studying the first two vitamins is that they play a key metabolic role in supplying methyl groups for DNA methylation in tissues. In addition, recent studies suggest that poor dietary intake of folate, particularly when combined with other breast cancer risk factors such as alcohol, may increase the risk for breast cancer. Our study design proposes to collect 100 primary breast carcinomas from women who have not been on estrogen replacement therapy. A sample of adjacent uninvolved breast tissue will be obtained from each breast carcinoma specimen. In Specific Aim 1, fresh frozen breast tissues will be used to evaluate differences between breast carcinoma and adjacent normal breast tissue, with respect to DNA methylation (global, anti-5-methylcytosine, CpG island and gene specific). In Specific Aim 2, the focus will shift to the relationship between tissue, circulating and dietary folate, vitamin B12 and vitamin C status, DNA methylation and MTHFR polymorphism in these breast cancer patients and controls. Our proposal will have an impact on breast cancer because it will provide basic information relative to factors that control methylation of specific genes or regions of the chromosome that are important in the development of breast cancer. DNA hypomethylation in cancer, as well as chromosomal alterations that may result from hypomethylation, have been known for some time. These changes in chromatin structure could play a very important role in the development of breast cancer, but they have been largely uninvestigated. We propose to fill this gap in knowledge by investigating the association between gene-specific and global DNA methylation patterns and breast cancer.